Fragile X Syndrome (FXS) is the most common inherited form of mental insufficiency and most prevalent monogenic cause of autism, occurring in ~1 in 4,000 males and ~1 in 5,000 females. The disease is caused by a CGG repeat expansion in the 5' untranslated region of the X-linked FMR1 gene that results in epigenetic silencing of FMR1. As a consequence, the product of FMR1, the fragile X mental retardation protein (FMRP), is not produced. FMRP is an RNA-binding protein that normally represses mRNA translation in the brain; in its absence, protein synthesis is excessive, which results in disease pathology. Reactivation of epigenetically silenced FMR1 is a promising new therapeutic approach for FXS that aims to correct the root cause of the disease rather than a secondary, downstream consequence of the FMRP deficiency. In preliminary experiments my laboratory has performed a small-scale candidate-based screen to identify eight repressive epigenetic regulators that promote silencing of FMR1 in FXS cells (called FMR1 Silencing Factors, or FMR1- SFs). Inhibition of FMR1-SFs by short hairpin RNAs (shRNAs) or small molecules reactivates epigenetically silenced FMR1 in undifferentiated induced pluripotent stem cells, neural progenitor cells, and post-mitotic neurons derived from FXS patients. These preliminary results provide important proof-of-concept regarding the feasibility of reactivating the epigenetically silenced FMR1 gene as a therapeutic approach for FXS. In this application we propose experiments using large-scale candidate-based and unbiased loss-of-function screens to identify additional FMR1-SFs, some of which may provide more desirable targets for the development of drugs that function by reactivating FMR1. To determine whether the level of FMR1 reactivation obtained with shRNA and small molecule FMR1-SF inhibitors is likely to be therapeutic, we will undertake several complementary approaches. First, we will determine the minimal level of FMRP required to normalize the well- characterized transcriptional, translational and morphological abnormalities of FXS neurons. In these experiments we will ectopically express FMRP at varying levels and analyze the effect on FXS neuronal phenotypes. Second, we will determine whether the level of FMR1 reactivation we obtain with shRNA and small molecule inhibitors of the FMR1-SFs we identify is sufficient to normalize dysfunctional phenotypes of human FXS neurons. In summary, the results of the proposed experiments will: (1) identify new targets whose inhibition leads to FMR1 reactivation, (2) determine the minimal levels of FMRP required to correct FXS neuronal dysfunctions, and (3) test whether FMR1 reactivation by shRNA and small molecule FMR1-SF inhibitors can normalize dysfunctional phenotypes of human FXS neurons. We believe the results of the experiments proposed in this application will have a major impact on the field of FXS therapeutics and have the potential to lead to development of a new class of drugs that can ameliorate this devastating disease.